Abstract
The molecular mechanisms that control the targeting of newly synthesized G protein-coupled receptors (GPCRs) to the functional destinations remain poorly elucidated. Here, we have determined the role of Golgi-localized, γ-adaptin ear domain homology, ADP ribosylation factor-binding proteins 1 and 2 (GGA1 and GGA2) in the cell surface transport of α2B-adrenergic receptor (α2B-AR), a prototypic GPCR, and studied the underlying mechanisms. We demonstrated that knockdown of GGA1 and GGA2 by shRNA and siRNA significantly reduced the cell surface expression of inducibly expressed α2B-AR and arrested the receptor in the perinuclear region. Knockdown of each GGA markedly inhibited the dendritic expression of α2B-AR in primary cortical neurons. Consistently, depleting GGA1 and GGA2 attenuated receptor-mediated signal transduction measured as ERK1/2 activation and cAMP inhibition. Although full length α2B-AR associated with GGA2 but not GGA1, its third intracellular loop was found to directly interact with both GGA1 and GGA2. More interestingly, further mapping of interaction domains showed that the GGA1 hinge region and the GGA2 GAE domain bound to multiple subdomains of the loop. These studies have identified an important function and revealed novel mechanisms of the GGA family proteins in the forward trafficking of a cell surface GPCR.
Highlights
G protein-coupled receptors (GPCRs) are the largest superfamily of cell surface receptors and their functions are highly regulated by intracellular trafficking processes
We have recently demonstrated that GGA3 is required for the trans-Golgi network (TGN)-to-cell surface transport of α2B-adrenergic receptor (α2B-AR), a prototypic member of the GPCR superfamily, and that the function of GGA3 in modulating α2B-AR export is mediated through its VHS domain interaction with the receptor, providing the first evidence implicating a role of the GGA family proteins in GPCR trafficking[47]
HEK293 cells were transfected with previously characterized shRNAs targeting GGA1 and GGA2 (Fig. 1A) and the effect of depleting individual GGAs on the numbers of α2B-AR at the cell surface was quantified by intact cell ligand binding assays using the cell nonpermeable radioligand [3H]RX821002 after doxycycline induction for different time periods. shRNA-mediated knockdown of GGA1 and
Summary
G protein-coupled receptors (GPCRs) are the largest superfamily of cell surface receptors and their functions are highly regulated by intracellular trafficking processes. Our laboratory is interested in dissecting the mechanisms of anterograde trafficking of GPCRs. We have recently demonstrated that GGA3 is required for the TGN-to-cell surface transport of α2B-adrenergic receptor (α2B-AR), a prototypic member of the GPCR superfamily, and that the function of GGA3 in modulating α2B-AR export is mediated through its VHS domain interaction with the receptor, providing the first evidence implicating a role of the GGA family proteins in GPCR trafficking[47]. We have found that all three GGAs are important in regulating the cell surface export of α2B-AR and more interestingly, three GGAs physically associate with the receptor via distinct domains These studies have revealed novel mechanisms of the GGA-mediated cell surface GPCR trafficking
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